Image Is Everything

Date: February 1, 1999Table of Confocal Microscope Manufacturers Perhaps in few other fields has the creation of an instrument been so important to the establishment of a new theory or discipline. Even the Galilean telescope, with its revelation of the Medicean moons, does not compare to the microscope because the foundation for astronomy had already been well established by naked-eye observation. Cell theory, by contrast, had no such foundation in anecdotal experience. However, it wasn't long

Feb 1, 1999

Brent Johnson

Perhaps in few other fields has the creation of an instrument been so important to the establishment of a new theory or discipline. Even the Galilean telescope, with its revelation of the Medicean moons, does not compare to the microscope because the foundation for astronomy had already been well established by naked-eye observation. Cell theory, by contrast, had no such foundation in anecdotal experience. However, it wasn't long before the theoretical limits of the compound microscope had been reached by the manufacturer Carl Zeiss in 1880.1 His magnification of 2,000 diameters and high resolution of about 0.2 µm, attainable by oil immersion, would not be significantly improved upon until the 1940s.

It seemed as though microscopy for the purposes of biological investigation had reached an impasse. Then in 1955, a new postdoctoral fellow at Harvard began applying his considerable talents to the problem of brain function. He considered methods for analyzing neurons in situ and developed a microscope that would resolve only the subject of interest rather than the surrounding tissue. He achieved this result by introducing a pinhole aperture in opposition to the focal lens of a light microscope. The effect of the pinhole aperture was to collect light that was returning from the focus point while eliminating scattered light that corrupts most typical microscopic images. However, the problem with this arrangement is that only small, pinhole-sized, images can be resolved at a given time. The solution to this difficulty was to move his sample underneath the imaging spot. By deflecting the platform vertically and horizontally with a magnetic solenoid he was able to produce a scanning pattern.2 The idea of stage scanning persisted even with some of Meridian Instruments' early systems. These microscopes moved the specimen in X,Y or rastered the laser with a galvo mirror in one axis and moved the stage in the orthogonal. Nevertheless, upon making this discovery, he quietly filed a patent application and returned to his work on artificial intelligence that has made Marvin Minsky one of the popular celebrities of science. Likely, Minksy never profited from his invention because his patent had expired by the time the laser and the practical computer were available. It was not until some time later that Davidovits and Egger were able to reconstruct Minsky's pioneering effort with their own original discovery.3

From the days of these early forays into confocal systems it has been discovered that the confocal microscope may yield yet another significant advantage in addition to removing extraneous light from the emission signal. It has been theorized that some samples survive longer under point illumination. Jim B. Pawley, a professor of zoology and editor of the standard reference, Handbook of Biological Confocal Microscopy, has suggested that electrons in the fluorochrome under full illumination are driven into higher orbitals and thus do not participate in fluorescence, making them vulnerable to ionization. A spot scanner would be a superior innovation in respect of the fact that each pixel is illuminated less than 1/(512x512th) of the time, allowing the fluorophor a chance to relax back to its unexcited state. In view of the demonstrated advantages that confocal technology has to offer, a number of companies have recognized the commercial potential of this field. What follows is an alphabetical listing of confocal manufacturers.

David Jaffe first became involved with confocal microscopes in 1992 while conducting research in the laboratory of Thomas Brown at Yale. Then, as a postdoc, and today, as a professor and lecturer on the principles of confocal microscopy at the University of Texas, San Antonio, Jaffe has used Bio-Rad microscopes almost exclusively. In fact, he insisted on getting one for his confocal laboratory for the reason that it is easy to use and generally reliable.

"The interface was very nice for students. They don't have to touch the hardware, the laser, the scanner, etc.... Controlling everything from the software end was key," said Jaffe.

Reflecting Jaffe's comments, John Jordan, who is the product manager for the Hercules, Calif. based company, emphasized how Bio-Rad is concentrating on producing easier-to-use interfaces so that the typical user needs to know less and less about the details of collecting a good image.

One of the most important new designs in the field of laser scanning microscopy is Bio-Rad's new multiphoton microscope. The primary benefit of multiphoton or two-photon microscopy is that it can penetrate much deeper into tissue samples, permitting an unobstructed view of cells such as neurons. The way it accomplishes this feat involves an unusual characteristic of light. When two photons hit a fluorophor simultaneously, it's as if one photon of half the wavelength excited the targeted fluorophor. If two photons of infrared light are directed at a particular sample, they will penetrate to a depth of several millimeters and then combine in the focal plane where the photons are most concentrated, reacting as if one photon of blue light hit the sample. The result is a deep tissue image of superior resolution. Although confocal and multiphoton techniques are generally considered to be overlapping technologies, some enthusiastic supporters consider the multiphoton technique a replacement for confocal microscopy.

Dr. Thomas U.L. Biber, director of the Confocal Microscope Facility at Virginia Commonwealth University, has been working with several teams of researchers. They are impressed by the outstanding optical perfomance and versatility of the Zeiss LSM 410 Confocal Microscope at the facility. The capability of the confocal microscope to obtain very thin optical sections of tissue samples and living cells at the highest resolution has proven to be extremely useful for many different applications such as 3-D reconstructions of cells and tissues, measurements of changes in cell volume and intracellular calcium activity as a function of time, and localization of antibodies within cells. Thus it was possible to test the colocalization of three different antibodies in subcellular structures of the same cell by labeling the antibodies with fluorescence in three different wavelengths (ultraviolet, green, and red).

Biber collaborated on a project conducted by Dr. E.R. Jakoi and coworkers. The project is concerned with the problem of regulation of localized protein synthesis within neurons and centers on the targeting of mRNA and mRNA binding proteins to special locations within hippocampal neurons. The investigation involves a variety of techniques such as treatment with antisense oligonucleotides, fluorescence in situ hybridization, and injection of minute amounts of compounds with fluorescence labels into living cultured cells. Confocal microscopy was needed to obtain a series of images for following the movement of fluorescent compounds to particular locations. For several reasons, the very good efficiency (i.e. high image intensity per laser power) of the Zeiss LSM 410 was very helpful in these experiments: First, the laser power could be decreased to a level which minimized the fading of the fluorescence due to photobleaching and, in addition, reduced the damage inflicted by the laser light to the living cells. Second, the optical sections of the cells could be made very thin ( about 0.6 microns thick) which on one hand improved the definition of the subcellular structures and on the other hand decreased the halo formation caused by fluorescence. Third, the image quality could be improved by using slower scans and/or by averaging several pictures.

Buddy Bossmann, the product marketing manager for Carl Zeiss' confocal microscope group based in Thornwood N.Y., explained that the latest generation LSM (the LSM 510) continues where the LSM 410 leaves off. The LSM 510 provides improved optical efficiency, higher sensitivity, and increased resolution by incorporating a shorter light path, 12-bit digitization, and an image size up to 2K by 2K pixels. In addition, Zeiss has just recently introduced a version of the LSM, the LSM 510 NLO (Non Linear Optics) that is multi-photon capable.

Leica Microsystems is the manufacturer of the TCS NT confocal system. Its significant advances in confocal technology have not been made at the expense of wide applicability and general utility. One of the significant modifications that Leica has made to this instrument is the invention of a merge module that extends multiline laser operation by connecting up to four different lasers. The TCS NT also has a UV fiber coupling that is designed to keep heat and vibration away from the microscope.

According to Christian Kier, technology manager for Leica Microsystems Imaging Systems Division in Exton Pa, the latest model TCS SP is an innovation in confocal microscopy. It allows for filterless detection over any spectral range. This translates into optimal spectral separation during simultaneous detection of multiple fluorescent probes, and custom detection of any fluorescent probe. Additionally, the TCS SP has inherently better light transmission and spectral response than a conventional filter-based confocal microscope leading to sharper, brighter images. All these strengths ultimately lead to greater experimental freedom for the investigator.

Molecular Dynamics which operates out of Sunnyvale, Calif. entered the microscope industry with the acquisition of Sarastro in 1990. At the time, the company was dealing with general purpose, research-grade upright and inverted microscopes. However, with the latest advances in confocal technology, confocal optics have become a common thread that has unified all of the company's instrumentation. The Sarastro 2000 and Multiprobe 2010 CLSM, the MegaBASE® DNA sequencer, STORM® imager and the new Avalanche® microscanner all employ confocal optics for fluorescence detection. While the Avalanche scanner was designed for scanning microarrays, it is also the first inherently quantitative confocal microscope. Taken together, these instruments serve as the platform for a variety of expandable features and adaptations.

Dr. Rick Rogers, who has been using confocal microscopes since 1989 created The BioMedical Imaging Lab at the Harvard School of Public Health in 1991. When Rogers was looking for instrumentation to equip his laboratory, he looked very carefully at software and computer platforms and chose the Molecular Dynamics' instrument primarily because of its software product called ImageSpace, which runs on a Silicon Graphics Unix-based platform.

"It is extremely powerful, fast, and seamlessly handles large volumes of image data," said Rogers, who archives up to a gigabyte of data every week and currently uses confocal microscopes manufactured by Molecular Dynamics, Leica, annd Zeiss. Although the ImageSpace software that he evaluated in 1991 has changed over the years, he still considers it to be on the forefront of data acquisition software and analysis.

The Nikon PCM 2000 is priced to fall in the category of instruments that a scientist can buy on an individual grant. A PCM-2000 with two lasers and two photomultipliers sells for about $100,000.

Edward Ziff, who is a Professor of Biochemistry and Investigator of the Howard Hughes Institute at the New York School of Medicine, has been using a Nikon PCM 2000 confocal microscope for about the last five months. Ziff's instrument has both Argon ion and HeNe lasers and is mounted on an Eclipse 800 microscope. It cost $140,000, including the E-800, however, this is still $100,000 cheaper than some comparable instruments. Ziff has found the microscope to be extremely useful in localizing receptors in brain sections, as well as in working out the morphology of individual cells.

"We make measurements on cells we had never considered," said Ziff. And because the instrument is located within his laboratory, its convenient access allows it to run over five hours a day producing images for their research. Ziff is considering the purchase of an additional laser. He does a great deal of fluorescein and rhodamine comparison, and having a HeNe laser at 633 nm would enhance the range of available fluorophors he can use.

Jeff Larson, who is the product manager for Nikon's confocal microscope division which is headquartered in Melville, N. Y., explained that the Personal Confocal Microscope is the culmination of three years of major fine-tuning of the software and minor fine-tuning of the microscope. The result is a simplified confocal microscope that features a solution to the problem of information overload. The PCM has two monitors which separate the interactive control display from the viewing screen. This configuration allows the researcher to manage data while observing the sample image simultaneously. The PCM also scans and images through a single pinhole. This does away with the necessity for delicate alignment and makes it possible to easily switch the PCM scanning head between microscopes. Images in both channels are collected and displayed simultaneously assuring exact temporal and spatial registration between them.

According to Mike Ignatius of Technical Instruments, which is a distributor of the Nikon PCM, the future of confocal microscopy is improved photon sensitivity during image capture and deconvolution of acquired images. Currently, photomultipliers are only about 30 percent photon efficient. Yet through a strategic alliance, Technical Instruments is going to begin packaging its Nikon PCM with Applied Precision's Delta Vision Image Restoration and Deconvolution System which improves light sensitivity while adding the capacity to deconvolve the image. The combined package will be offered for $300,000 and will resolve approximately 0.2 micron in five different colors. This allows the option of doing deconvolution and confocal on one platform.

Noran Instruments in Middleton, Wisconsin has developed a confocal system that is intended for use by researchers who are specifically interested in live action. By integrating a Noran Acoustic Optical Deflector with an ultrafast piezoelectric objective lens translator and Silicon Graphics imaging technology, Noran has produced the fastest confocal scanner available.

"Most confocal scanners don't scan fast enough for live samples," explained Adam Myerov, product manager for Noran. However, at a scanning range that starts at 30 frames per second (the normal speed of a video camera) and extends as high as 480 frames per second, live fast scanning is what Myerov appropriately describes as Noran's "bread and butter."

Scott Pomeroy has used a Noran confocal microscope for the past two years in his work at Children's Hospital in Boston. In addition to the fast scan rate, which he considers to be among the best, Pomeroy believes the fundamental value of Noran confocal microscopes is their ability to obtain optical sections that are otherwise hidden by structural overlay. The automatic sectioning of samples into three dimensions has saved countless hours of manually sectioning tissue, and it preserves the sample intact.

Olympus America in Melville, N.Y. provides the Fluoview confocal microscope which has been designed with ease of use in mind. This instrument employs a number of features such as one-touch tag menu selection and an auto gain function which permits users to image samples without making complicated adjustments. Features like these have made the Fluoview one of the bedrock instruments at the Bakewell Neuro-Imaging Laboratory where director Jeff Lichtman is a professor of neurobiology. Lichtman has had a wide experience of using confocal instruments. In fact, his 1989 patents of a spinning disk confocal microscope anticipated some of the more recent Nipkow disk units which are beginning to catch on in popularity.

For the past two years Lichtman has been using an Olympus Fluoview to image synapses in living mice. The groundwork for his discoveries about the function of the nervous system were made with the Olympus Fluoview, the Bio-Rad MRC 1024, and the Noran Odyssey. What Lichtman likes particularly about the Olympus is its software package which allows users to get images without extensive training.

"It is relatively inexpensive and yet, it has most of the features you would like to see," said Lichtman. "Users who don't have a great deal of sophistication about how to acquire images or generate data can use this device from beginning to end."

In addition to the multiphoton confocal, one new design that is making a profound impression on those who have used it is the Yokogawa CSU-10 from Yokogawa Electric Company in Japan. This is a direct view microscope that attains speeds of 6 ms per frame or 1 ms with a special motor. The unit itself attaches to any inverted or upright microscope, and for a mere $50-60,000 it is one of the least expensive confocals in the industry. Only the Noran confocal shares the speed benefits of the Yokogawa and the flexibility of point scanning that make these instruments attractive for a wide variety of applications.

According to Ted Inoue, president of Universal Imaging and one of the major believers in the new technology, what makes the Yokogawa unit unique is that it functions by focusing a laser onto a disk containing 20,000 pinholes. Each pinhole is imaged onto the specimen with the aid of microlenses that help to collect more light from the laser, resolving one of the shortcomings of previous attempts at making this a workable design. As the pinhole disk spins it paints a swath of light across the sample, covering each point with an equal amount of light. Yokogawa's patented pinhole array ensures even illumination across the entire sample.

Some drawbacks to the new design have to do with its fixed scanning pattern. This makes the instrument undesirable for scanning single lines or points. In addition, the size of the pinholes are fixed, resulting in optimal confocality with only specific objective magnifications. Despite these minor problems, however, some authorities are hailing the Yokogawa CSU-10 as a major achievement.

"It is quite an awesome invention," said Shinya Inoue, who is the senior author of Video Microscopy and has been described as the grandfather of modern light microscopy. Inoue, who has been a beta user for the instrument, is enthusiastic about this new design. He explained his surprise as the Yokogawa unit imaged even some very difficult samples such as some sparsely distributed 20 nm fluorescent spheres.

"The Yokogawa unit was easy to use and had no problem seeing the samples," said Inoue.

Perhaps one of the most compelling features of the Yokogawa is its ease of use. It has virtually no adjustments so it's well suited for the multi-user facility or student training. The Yokogawa CSU-10 made one of its first public appearances at Neuroscience 97 and has been commercially available for about the last few months.

In a field where scientists had become pessimistic about the possibility for improving the resolution of microorganisms, the confocal microscope has offered some important new discoveries. Although the confocal microscope may never entirely replace light microscopes or electron microscopes, it does fill an important niche in revealing live motion and three-dimensional structures. As progress continues to be made with data acquisition software and fluorescent dyes as well as new design ideas such as the multiphoton microscope and the acoustic optical deflector, confocal microscopy will undoubtedly serve an increasing role in fundamental research.